JP4711846B2 - 方法 How to determine acute mastitis - Google Patents

Description

  The present invention relates to a method for easily and quickly determining whether bovine mastitis is acute acute mastitis and a tool therefor.

  Mastitis accounts for the highest number of dairy cattle hurts, and the economic losses from mastitis are enormous. Mastitis is a disease caused by inflammation of the mammary gland tissue and is caused by various stimuli such as microbial infection, their toxins, physical trauma, etc., but the main cause is microbial infection, among which infection by pathogenic bacteria is mostly It is. Symptomatic clinical mastitis is generally classified into epilepsy acute mastitis, acute mastitis, and chronic mastitis. Of these, epilepsy mastitis is only a few hours to a day. It is the most dangerous and serious mastitis because there are many cases that lead to fatalities with rapid deterioration of symptoms.

  原因 The causative bacteria of acute mastitis are mainly enterobacteria, and most of them are infected by microorganisms belonging to the genus Escherichia coli and Klebsiella. Since infection with these microorganisms causes endotoxin shock, it is necessary to diagnose whether cattle with mastitis have acute acute mastitis at an early stage, and there is a strong suspicion of acute acute mastitis In some cases, it is necessary to promptly take appropriate treatments such as endotoxin excretion and administration of antibacterial agents (for bovine mastitis, the “Guidelines for Livestock Mutual Aid II”, National Agricultural Mutual Aid Association, Issued in 1985, see 5. Guidelines for Mastitis Treatment (pp.178-313)).

  Conventionally, in order to identify the causative agent of mastitis, a sample such as milk is collected and cultured on an agar medium, and the type of the fungus is identified by the biochemical properties of the colony or Gram staining Is adopted. However, this method usually requires 1 to 3 days of culture at 37 ° C., and treatment of acute mastitis is often too late. In addition, it may be possible to diagnose early acute mastitis by direct smear of milk and Gram staining, but it is difficult to finally identify the bacteria from the shape of the bacteria, Since staining and observation under a microscope are necessary, there is a problem that it cannot be adopted as a rapid diagnostic method in the dairy field. Furthermore, although various media and identification kits are provided for bacteriological examination, none of them is satisfactory from the viewpoint of rapidity and simplicity. Under such circumstances, it is desired to develop a method that allows dairymen and veterinarians to quickly identify the causative bacteria of mastitis on site, and in particular, E. coli and Klebsiella, which are the main causative bacteria in acute acute mastitis, are rapidly developed. The development of a method that can be easily specified and a simple tool is desired.

On the other hand, the indole test, which is widely used as one of the methods for identifying microorganisms, is a method for examining the presence of indole generated by tryptophan degradation by adding a coloring reagent. . The VP test (Voges-Proskauer test) is a method for detecting acetylmethyl carbinol produced under acidic conditions from two molecules of pyruvic acid generated by the degradation of glucose. Since the production of acetylmethyl carbinol is extremely characteristic depending on the bacterial species, it is widely used as a method for distinguishing intestinal bacteria such as Klebsiella (“testing method for enteric pathogens”, Hiroshi Zenyoji et al. Written by Medical Shoin Co., Ltd., 1985, 4th edition, pp.24-33 and pp.73-76
Medical Care Mutual Aid II, National Agricultural Mutual Aid Association, Revised in 1985, pp.178-313 Test method for intestinal pathogens, written by Hiroshi Zenyoji et al., Medical Shoin Co., Ltd., published in 4th edition, 1985, pp. 24-33 and pp. 73-76

  An object of the present invention is to provide a method for quickly and easily determining whether bovine mastitis is acute acute mastitis and a simple tool for the method.

  The present inventor found that most of acute acute mastitis is caused by microorganisms belonging to the genus Escherichia coli and Klebsiella, and when the above microorganisms are present in an infected sample, the acute acute mastitis develops with a high probability. Based on the facts, intensive research was conducted to provide a method for quickly and easily confirming the presence of the microorganisms in infected samples. As a result, since bovine milk contains tryptophan and lactose, if the causative bacterium of mastitis is Escherichia coli, indole is contained in the milk collected from the bovine or in the mastitis sample. When the causative agent of the flame is a microorganism belonging to the genus Klebsiella, it has been found that acetylmethyl carbinol is contained in the milk or the mastitis sample. In addition, by confirming whether or not indole and / or acetylmethyl carbinol is contained in milk or mastitis samples, the bovine individual can undergo acute acute mastitis without performing microorganism identification operations such as culture. The present inventors have found that it is possible to easily determine whether or not symptom has developed with high accuracy and that a method using a capillary column is suitable from the viewpoints of simplicity and speed. The present invention has been completed based on the above findings.

That is, according to the present invention, a method for determining whether the mastitis of a bovine individual who developed mastitis is acute mastitis, wherein indole and mastitis are collected in milk or mastitis sample collected from the bovine individual. If acetylmethyl carbinol is included, a method is provided for determining that the bovine individual is likely to develop epilepsy mastitis.
The method described above directly demonstrates the presence of indole and / or acetylmethyl carbinol contained in milk or in a mastitis sample without performing a microbiological culture procedure using the milk sample. This is a method for determining that an individual has a high possibility of developing acute mastitis.
According to the present invention, there is also provided a method for diagnosing cattle acute mastitis, comprising the step of measuring indole and / or acetylmethyl carbinol contained in milk or mastitis sample collected from the bovine individual. A method is provided.

  From another point of view, it is a tool for determining whether a bovine individual who has developed mastitis has acute mastitis, comprising a chromatographic carrier in a capillary tube, and milk collected from the bovine individual Alternatively, when a sample of mastitis is sucked into the capillary tube from one end of the capillary, and then a coloring reagent solution for indole and / or acetylmethylcarbinol is sucked from the same end, and color development by the coloring reagent is observed. Provides a tool for determining that the mastitis is likely to be acute mastitis.

  With the method and tool of the present invention, it can be determined very simply and quickly whether or not the mastitis developed by a bovine individual is acute mastitis, and there is no need to perform a complicated and long-term culture operation, The determination can be performed in a very short time using the milk or mastitis sample as it is. If it is determined to be acute mastitis, appropriate treatment by a veterinarian can be immediately performed.

  The method of the present invention is a method for determining whether the mastitis of a bovine individual who has developed mastitis is acute mastitis, wherein indole and mastitis are collected in a milk or mastitis sample collected from the bovine individual. In the case where acetylmethyl carbinol is contained, it is characterized by including a step of determining that the bovine individual is highly likely to develop acute mastitis. Milk collected from bovine individuals may be obtained by normal milking, but preferably as sterilely and hygienically as possible to avoid misjudgment (e.g. avoiding contamination such as manure) ) It is desirable to use a collected sample. In the method of the present invention, in addition to a milk sample, a mastitis sample such as a tissue fluid, a pus-like sample, or a coagulum from a mastitis breast can also be used. The sample is preferably used immediately after being collected without being stored, but a sample that has been refrigerated or frozen may be used. The sample may be diluted as appropriate with an appropriate aqueous medium, for example, water, buffer solution, physiological saline or the like. In the case of using mud milk, the supernatant may be used as a sample by performing centrifugation after diluting with the above-mentioned aqueous medium and allowing to stand as necessary. Moreover, it is also preferable to use whey (whey) from which milk is allowed to stand in a cold place and the solid content is removed as a sample.

  The indole test and the VP test for detecting acetylmethyl carbinol are widely used by those skilled in the art. For these reactions, a reagent that generally reacts with indole and acetylmethyl carbinol (also called acetoin) to develop color. Can be used. As the indole reagent, for example, p-dimethylaminobenzaldehyde can be used. In the VP test, for example, α-naphthol can be allowed to react under basic conditions and then reacted with a guanidine source (for example, creatine). A solution containing α-naphthol and a guanidine source may be used as a reagent. However, the reaction reagent is not limited to these, and any reagent may be used as long as it can detect indole and acetylmethylcarbinol by color development. The details of the indole test and the VP test are described specifically and in detail in Non-Patent Document 2, for example, and those skilled in the art can easily perform these tests based on this description.

  Among microorganisms belonging to the genus Klebsiella, microorganisms that can be detected by the VP test include, for example, Klebsiella oxytoca, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella terigena (rigleella rig) , Klebsiella trevisanii and the like, but is not limited thereto.

  It can be easily diagnosed by a dairy farmer or a veterinarian that a bovine individual develops mastitis, usually by systemic symptoms such as body temperature, pulse, breathing, and examination and palpation of the breast and nipple. However, it is often difficult to determine whether the mastitis is acute mastitis or acute or chronic mastitis until a bacteriological test is performed. In particular, it is usually very difficult to make a diagnosis by distinguishing between acute mastitis and acute mastitis without bacteriological examination.

  The method of the present invention focuses on the fact that most of the causative bacteria of acute mastitis are microorganisms belonging to Escherichia coli or Klebsiella, and when these microorganisms are present in an infected sample, the sample is indole test and It was completed based on the knowledge that a positive result was obtained in the VP test. If a positive result was obtained in these tests, it was judged that there was a strong suspicion of infection by microorganisms belonging to Escherichia coli or Klebsiella. However, it is characterized in that it is possible to determine that the possibility of acute mastitis is high. Then, after it is determined that the possibility is high, it is possible to immediately start appropriate treatment such as detoxification treatment for endotoxin and administration of an antibacterial agent for infection. When culturing Escherichia coli producing indole in a medium containing tryptophan (such as LIM medium), if the medium is prepared using milk (commercially prepared milk, etc., commercially available ingredients) and cultured overnight, the indole is usually almost all Not produced and undetectable. On the other hand, the reason that the presence of indole can be proved directly from a milk sample or mastitis sample according to the method of the present invention is that E. coli infection occurs near the exit of the mammary gland, and a large amount of E. coli comes into contact with the secreted milk containing concentrated tryptophan. It is believed that this is because a concentration of indole is produced that is contaminated in the milk or mastitis sample. When performing the VP test, it is particularly preferable to use whey instead of the milk or mastitis sample.

  The method of the present invention can be carried out more quickly and easily by using the tool provided by the present invention. The device of the present invention is a device for determining whether or not a bovine individual who has developed mastitis has acute mastitis, comprising a chromatography carrier in a capillary tube, and milk collected from the bovine individual Alternatively, when a sample of mastitis is sucked into the capillary tube from one end of the capillary, and then a coloring reagent solution for indole and / or acetylmethylcarbinol is sucked from the same end, and color development by the coloring reagent is observed. Is a tool for determining that the mastitis is likely to be acute mastitis.

  As the capillary, for example, a capillary having an outer diameter of about 2 to 5 mm, preferably about 3 to 4 mm, and an inner diameter of 1 to 3 mm, preferably about 2 mm can be used. The inner diameter of both ends of the capillary is preferably narrowed, and the tip is particularly preferably an inner diameter of about 1 to 2 mm, preferably about 1 mm, but the suction part also has the effect of filtering the sample Therefore, it is also preferable to make the diameter larger than the outer diameter of the capillary. The shape of the suction part is not particularly limited, and can be appropriately selected according to the purpose and desired performance. The length of the capillary is not particularly limited, but is, for example, about 50 to 200 mm, preferably about 100 to 150 mm. The material of the capillary is not particularly limited as long as it is a transparent member such as glass or acrylic, and glass is generally preferable. As the chromatography carrier, for example, silica gel, polyamide, or alumina for column chromatography can be used, and polyamide is preferred. The particle size of the carrier is not particularly limited, but is usually in the range of about 100 to 500 μm. For example, a polyamide suitable for the device of the present invention is sold by Wako Pure Chemical Industries, Ltd. as polyamide C-100 (particle size 150-425 μm).

  When a glass capillary having a total length of about 120 mm, an outer diameter of 3.4 mm, and an inner diameter of about 2 mm is used as the tool of the present invention, for example, the inner diameter of both ends is narrowed to about 1 mm with a flame, and a chromatography carrier stopper is used. For example, absorbent cotton can be filled with about 2 to 10 mm, preferably about 3 to 5 mm, and then a chromatography carrier (for example, polyamide) can be filled with about 10 to 30 mm, preferably about 20 mm. One or several glass beads may be used as the stopper, but the stopper is not limited to absorbent cotton or glass beads, and a stopper that prevents the packed chromatography carrier from leaking out may be appropriately selected. The packing amount of the chromatography carrier is not particularly limited, but is, for example, about 20 to 50 μl, preferably about 30 to 40 μl. After filling the carrier, absorbent cotton can be further filled with about 2 to 10 mm, preferably about 3 to 5 mm, to form a carrier stopper.

  One end of the above tool is connected to a syringe or the like, and the milk or mastitis sample is sucked from the other end. The amount of sample suction is not particularly limited, but it is usually about several tens of μml to several ml, and it is preferable to suck this amount of sample into the capillary tube over several seconds to several minutes. After aspirating the sample, a small amount of air is aspirated as necessary, followed by a solution containing indole reagent (eg, 5 g of p-dimethylbenzaldehyde, 10 ml of phosphoric acid, and 50 ml of methyl alcohol, or p-dimethylbenzaldehyde 5 g, a solution containing 25 ml of concentrated hydrochloric acid and 75 ml of normal (or iso) amyl alcohol) or a reagent for VP test (for example, liquid A: ethanol solution of 6% α-naphthol, and liquid B: creatine 0.3 g, hydroxylated A solution containing 40.0 g of potassium and 100 ml of purified water is prepared, and solution A and solution B are mixed in advance or at the time of use) or a reagent mixture thereof is sucked into the capillary tube.

  The amount of the reagent to be sucked is not particularly limited. However, when sucking the indole reagent, it is preferable to suck the amount that the entire chromatographic carrier is immersed. When polyamide is used as a chromatographic carrier, indole is strongly adsorbed on the polyamide, and reacts with the reagent on the carrier to develop color. When aspirating the reagent for VP test, aspirate the amount of the entire chromatographic carrier soaked in, then aspirate a small amount of air and extrude the aspirated reagent from the chromatographic carrier. It is preferable to leave the air layer at a position away from the carrier. Acetylmethyl carbinol is weakly adsorbed on the polyamide, and is eluted from the polyamide by suction of the reagent for VP test. By sucking a small amount of air, it moves into the reagent solution separated from the polyamide by the air layer. To do. In this reagent solution portion, the concentrated acetylmethyl carbinol and the reagent are sufficiently mixed, and the color development reaction proceeds efficiently to enable highly sensitive measurement. When a pus-like mastitis sample is used, acetylmethyl carbinol is adsorbed to the polyamide and may react with the reagent on the carrier to develop color. The amount of the VP test reagent to be sucked is not particularly limited, but it is preferable to suck the amount by which the entire chromatographic support is immersed.

If the concentration of indole or acetylmethyl carbinol in the sample is high, it can be judged by visual inspection immediately after the above suction, but if necessary, about indole reaction at room temperature or under heating at about 30 ℃ Is visually judged after being left for about 10 to 15 minutes, and for the VP test, it is visually judged for words left for about 15 minutes to 1 hour. When the indole reaction shows a slight red to red color, the VP test can be judged as positive when the slight red color changes to a red color, and when this color development is observed, the mastitis is acute mastitis It is determined that the possibility is high.

  Further, when carrying out the method of the present invention, a sample (for example, about 1 ml) is collected in a small test tube, and a chromatographic carrier such as polyamide is added thereto and stirred so that a certain amount can be sucked. For example, about 20 to 50 μl, preferably about 30 to 40 μl of the mixture obtained in a capillary tube lightly plugged with cotton at a position 2 cm from one end (length of the mixture part filled in the capillary) Is, for example, about 10 to 30 mm), and then, if necessary, a small amount of air is sucked, and further, an indole reagent, a VP test reagent, or a mixture thereof can be sucked. In this case, the length of the reagent part sucked into the capillary is, for example, about 4-5 mm.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.
Example 1
A glass capillary column (outer diameter: 3.40 mm, inner diameter: 1.90 mm, length: 120 mm) was prepared as follows. Both ends of the capillary were heated with a flame to an inner diameter of about 1 mm. By doing in this way, the cotton and glass beads of the stopper do not slide off, and there is no corner when connecting to a syringe for sample suction / injection, so operability is good. Lightly packed 3-5 mm from one end of the capillary using absorbent cotton as a stopper, and filled with polyamide (manufactured by Wako Pure Chemical Industries, Ltd., polyamide C-100, particle size 150-425 μm, 33 μl) so that there is no gap of about 20 mm. . Then, it was lightly packed about 3 to 5 mm using absorbent cotton as a stopper so that the polyamide would not spill.

  1 ml of milk collected from a bovine individual who developed mastitis was sucked at a suction speed of about 1 ml / min. Thereafter, an indole reagent (prepared from 5 g of p-dimethylbenzaldehyde, 25 ml of concentrated hydrochloric acid, and 75 ml of isoamyl alcohol) was sucked to immerse the polyamide part. Immediately after the suction and 15 minutes later, the color development was visually confirmed, and a slight red to red color was regarded as positive. As a result, a strong indole positivity was observed in milk collected from bovine individuals suspected of developing acute acute mastitis. From the milk collected from this bovine individual, Escherichia coli was identified by the subsequent microbiological test and staining test conducted in the usual manner, and it was confirmed that this bovine individual had developed acute mastitis. On the other hand, when milk collected from healthy cattle was used, no indole positivity was observed even after 15 minutes.

  In addition, when indole was added to commercial milk and the time required for color development was examined, 1 to 4 ml of sample containing 1 to 5 μg of indole was aspirated as described above. It was confirmed that color was developed. The reaction reached a peak in about 10 minutes, and the color gradually faded after 2 hours. Further, the sample containing 20 μg / ml indole gave a slight red color development in 30 seconds, and the color development peaked in about 10 minutes.

Example 2
The capillary column obtained by the method of Example 1 was aspirated with 0.5 ml of milk collected from a bovine individual who developed mastitis (a milk sample that had been stored frozen after sterility testing) at a suction rate of about 1 ml / min. Prepare VP test reagent (solution A: ethanol solution of 6% α-naphthol, and solution B: 0.3 g of creatine, 40.0 g of potassium hydroxide, and 100 ml of purified water. Suck the polyamide part by soaking, and then suck a small amount of air to expel the VP test reagent from the polyamide part, and leave the VP test reagent at a position about 5 mm away from the polyamide part. It was. Color development was visually confirmed after 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, and 3 hours. As a result, milk collected from bovine individuals suspected of developing acute acute mastitis showed VP positivity 15 minutes later. From the milk collected from this bovine individual, Klebsiella pneumoniae was identified by routine microbiological and staining tests, and it was confirmed that this bovine had developed acute mastitis. It was. On the other hand, when milk collected from healthy cattle was used, VP positivity was not observed even after 1 hour.

Example 3
Alternatively, 0.5 ml of the milk sample of Example 2 was placed in a small test tube having an outer diameter of 12 mm, an inner diameter of 9 mm, and a length of 105 mm, and VP reagent A solution and B solution were sequentially added. Color development was visually confirmed after 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, and 3 hours. As a result, the reaction was weakly observed at 120 minutes and positive at 180 minutes. In this method, the reaction took longer than the method of Example 2 using a column, and although the sensitivity was low, VP was not performed using milk collected from bovine individuals suspected of developing acute mastitis. It was judged positive.

Example 4
In a small test tube containing 50 μl of polyamide, 1 ml of the milk sample of Example 1 was added and mixed, and then allowed to stand to precipitate the polyamide. The supernatant was removed and the indole reagent was added dropwise to the polyamide in the test tube. A red color was observed on the polyamide after 15 minutes at room temperature.

Example 5
5 ml of the milk sample of Example 2 was placed in a small test tube containing 100 μl of polyamide and mixed, and then the polyamide was precipitated by centrifugation. The supernatant was removed, and the VP test reagent was added dropwise to the polyamide in the test tube. A red color was observed on the polyamide after standing at room temperature for 1 hour.

Claims (4)

A method mastitis in cattle individuals who developed mastitis determines whether there is a possibility of甚急mastitis due to infection of E. coli or Klebsiella pneumoniae, indole into the milk taken from the bovine individual If it is included, it is determined that the bovine individual is likely to develop acute mastitis due to infection with E. coli , and acetyl methyl carbinol is contained in the milk collected from the bovine individual. If you are a method of determining the bovine individual and are likely to be developed甚急mastitis due to infection of Klebsiella pneumoniae. A tool for determining whether mastitis in cattle individuals who developed mastitis is potentially甚急mastitis due to infection of E. coli or Klebsiella pneumoniae, a carrier for chromatography on a capillary tube The milk collected from the bovine individual is sucked into the capillary tube from one end of the capillary, and then the coloring reagent solution for indole or acetylmethyl carbinol is sucked from the same end, and the coloring by the coloring reagent for indole is developed. When it is recognized, it is determined that the mastitis is likely to be acute mastitis due to infection with E. coli, and when coloration by the coloring reagent for acetylmethylcarbinol is observed, the mastitis use for but be determined to be likely to be甚急mastitis due to infection of Klebsiella pneumoniae . A tool according to claim 2, wherein the chromatographic carrier is a polyamide. The tool according to claim 2 or 3, wherein the capillary is a glass capillary.